Alpha-galactosidase A (alpha-Gal A) is a lysosomal enzyme having an activity to hydrolyze the terminal alpha-galactosyl bond of glycolipids and glycoproteins. Trihexosylceramide, which contains three hexose moieties, is one of the substrates for alpha-galactosidase A and receives hydrolysis at its terminal hexose moiety by the enzyme. Human alpha-Gal A is expressed as a precursor peptide comprising 429 amino acids, of which N-terminal 31 amino acids constitute a signal peptide. The precursor is processed to the mature peptide, which consists of 398 amino acids left behind after the removal of the signal peptide, and a pair of the mature peptide molecules readily form a homodimer, wherever condition allows, which is the enzymatically active form of human alpha-Gal A. Human alpha-Gal A possesses oligosaccharide chains containing mannose-6-phosphate residues and can be targeted to the lysosome via a mannose-6-phosphate receptor.
Fabry's disease is an X-linked inherited lysosomal storage disease, which is caused by inherited abnormality of gene encoding alpha-Gal A. Lack of the activity of this enzyme causes accumulation of trihexosylceramide in various tissues, resulting in renal impairment, angiokeratomas, and cardiovascular abnormalities, including ventricular enlargement and mitral valve insufficiency.
It had already been known in the 1960's that the enzymatic activity of alpha-galactosidase A is scarcely or only weakly detected in the tissues of patients with Fabry's disease. It thus had been speculated that some inherited abnormality of the gene encoding alpha-Gal A should be the cause of Fabry's disease. Based on this speculation, alpha-Gal A replacement therapy for Fabry's disease had been conducted by infusing alpha-Gal A extracted and purified from human plasma or spleen to the patient, resulting in the confirmation that alpha-Gal A replacement therapy could lower the levels of trihexosylceramide in the blood of the patients suffering from Fabry's disease (see NPL 1, NPL 2, NPL 3). Therefore it had been considered that alpha-Gal A enzyme replacement therapy must be promising for Fabry's disease. Practical application of alpha-Gal A replacement therapy, however, has been extremely limited due to lack of supply of this enzyme.
In 1986, the gene encoding human alpha-Gal A was isolated (see NPL 4), and further, in 1988, a method was reported for expression and production of rh alpha-Gal A by using CHO cells transformed with a human alpha-Gal A expression vector (see NPL 5). Utilizing recombinant technology, these finding made it possible to produce human alpha-Gal A in a large scale, and to use the enzyme as a medicament for the enzyme replacement therapy for Fabry's disease. The rh alpha-Gal A has also been expressed in insect cells (PTL 1).
Further, some methods for purification of rh alpha-Gal A were reported comprising employment of hydrophobic column chromatography, heparin sepharose column chromatography, hydroxylapatite column chromatography, anion-exchange column chromatography, and gel filtration column chromatography (PTL 2, PTL 3). Although the purity of the rh alpha-Gal A obtained by the method for purification described in PTL 2 or PTL 3 has shown to be sufficiently high, it was not considered to be fully acceptable for human use. The reason is that while any medicaments to be infused to patients are required to be free of contaminants, such as, in particular, proteins derived from the host cells employed, either PTL 2 or PTL 3 fails to show whether such contaminants remain in the purified rh alpha-Gal A or not. And further a method for purification of rh alpha-Gal A were reported comprising employment of anion-exchange column chromatography, hydrophobic column chromatography, column chromatography employing as solid phase a material having affinity for phosphate group, cation-exchange column chromatography, and gel filtration column chromatography, in the order (PTL 4). However the PTLs are silent on contamination of rh alpha-Gal A with host cell proteins.
Currently, medical drugs for Fabry's disease containing rh alpha-Gal A as an active ingredient have been on the market under the trade names such as Replagal™ and Fabrazyme™.